Fluorometric reagents and methods

ABSTRACT

A novel class of reagents react with primary amine-containing compounds, preferably polypeptides and amino acids to form highly fluorescent products. This reaction serves as a basis for a rapid and highly sensitive assay method for primary amine-containing compounds.

United States Patent Leimgburger et al.

[111 3,871,825 1 Mar. 18, 1975 F LUOROMETRIC REAGENTS AND METHODSInventors: Willy Leimgburger, Montclair;

Manfred Weigele, North Caldwell,

both of NJ.

Assignee: Hoffmann La-Roche lnc., Nutley,

Filed: Mar. 4, 1974 Appl. No.: 447,610

Related US. Application Data Division of Ser. No. 212,790, Dec. 27,1971, Pat. No. 3,812,181.

US. Cl. 23/230 M, 23/230 B, 260/3433 lnt. Cl. G0ln 31/22, g01n 33/16Field of Search 23/230 M, 230 B;

[56] References Cited UNITED STATES PATENTS Udenfriend 23/230 M PrimaryExaminerRobert M. Reese Attorney, Agent, or Firm-Samuel L. Welt; Jon S.Saxe [57] ABSTRACT- 23 Claims, N0 Drawings F LUOROMETRIC REAGENTS ANDMETHODS This is a division of application Ser. No. 212,790, filed Dec.27, 1971, now US. Pat. No. 3,812,181.

BACKGROUND OF THE INVENTION The use of ninhydrin as a colorimetricreagent for the detection and assay of amino acids, amines and peptideshas been known in the art for nearly 60 years. It serves as the basisfor the well known Stein-Moore procedure now extensively utilized in theautomated assay of amino acids. McCaman and Robins described afluorometric method for the detection of serum phenylalanine involvingthe reaction between ninhydrin and phenylalanine and discovered thefluorescence was greatly enhanced by the addition of a variety ofpeptides. Later, Udenfriend and coworkers, Anal. Biochem. 42, 222, 237(1971), described a fluorometric assay procedure for primaryamine-containing compounds, especially peptides and amino acids, whichinvolved the reaction between the primary aminecontaining compound,ninhydrin and an aldehyde, preferably phenylacetaldehyde. This procedurewas shown to be much more efficient and sensitive than the Stein-Mooreand McCaman-Robins procedures.

BRIEF DESCRIPTION OF THE INVENTION The present invention relates to anovel series of compounds represented by formula I wherein R ishydrogen, halogen, lower alkyl or lower alkoxy and R is lower alkyl oraryl. These compounds produce highly fluorescent substances uponreaction with primary amine-containing compounds, and are thereforedesignated as fluorogens"v 1n the specification and the appended claims,the term "lower alkyl shall mean a monovalent, saturated, straight orbranched chain hydrocarbon substituent containing up to and including 8carbon atoms; the term lower alkoxy shall mean a group having a loweralkyl group linked to an ether oxygen and having its free valence bondfrom the ether oxygen; the term aryl shall mean an aromatic ring systemwhich may be substituted with one or more of the following: halogen(i.e., fluorine, chlorine, bromine or iodine), lower alkyl, loweralkoxy, nitro, cyano and so forth. Exemplary aromatic ring systemsinclude phenyl, naphthyl, furyl, thienyl, pyrrolyl, imidazolyl, pyridyl,pyrimidinyl, indolyl, oxazolyl, isoxazolyl, and so forth.

Preferred compounds of formula I are those where R is hydrogen. Examplesof preferred compounds of formula l are:

4-phenylspiro[furan-2( 3H ),1 '-phthalan]-3,3 '-dione 4-(2-methoxyphenyl)spiro[furan-2( 3H ),1 phthalan]-3,3-dione4-(3-methoxyphenyl)spiro[furan-2(3H),l phthalan]-3,3-dione4-(4-methoxyphenyl)spiro[furan-2( 3H ),1 phthalan]-3,3'-dione 42,4-dimethoxyphenyl)spiro[furan-2( 3H ),1 phthalan]-3,3-dione4-(2,5-dimethoxyphenyl)spiro[furan-2( 3H),l phthalan]-3,3'-dione4-(3,S-dimethoxyphenyl)spiro[furan phthalan]-3,3-dione4-(3,4,5-trimethoxyphenyl)spiro[furan-2( 3H ),1 phthalan]-3,3'-dione4-(2,4,5-trimethoxyphenyl)spiro[furan-2(3H), phthalan]-3,3-dione 4-(3,4-methylenedioxyphenyl)spiro[furan-2( 3H l phthalan]-3,3-dione 4-(3-chlorophenyl)spiro[furan-2( 3H ),1 '-phthalan 3,3'-dione4-(4-chlorophenyl)spiro[furan-2(3H ),l -phthalan 3,3-dio'ne4-(4-bromophenyl )spiro[furan-2( 3H ),1 phthalan]3,3'-dione4-(3-indolyl)spiro[furan-2( 3H l -phthalan]-3,3 dione4(Z-naphthyl)spiro[furan-2(3H),l '-phthalan ]-3,3 dione 4-(1-naphthyl)spiro[furan-2( 3H ),1 '-phthalan]-3,3 dione 4-( l-propyl)spiro[furan-2( 3H ),1 -phthalan ]-3,3 dione.

Especially preferred is the compound where R is phenyl, i.e.4-phenylspiro[furan-2(3H),l-phthalan]-3,3'- dione.

The preparation of compounds of formula I is illustrated in ReactionScheme A starting from compound 11. Compounds of formula 11 aregenerally known and are prepared starting with the properly substituted0- acetyl benzoic acid and the desired aldehyde R'CHO. Where particularmembers of the genus represented by formula II have not been previouslydescribed in the art, they may be prepared in the same manner as theknown ones.

In the first step, the enol lactone of formula II is hydrolyzed to theketo carboxylic acid of formula III by an aqueous basic hydrolysis,followed by acidification. Suitable bases forthe hydrolysis reactioninclude alkali metal hydroxides, e.g. sodium hydroxide, alkali metalcarbonates, e.g. sodium carbonate, and alkali metal bicarbonates, e.g.sodium bicarbonate. A preferred base is an alkali metal hydroxide suchas sodium hydroxide.

The temperature of the hydrolysis can be in a range of y from about 0 toabout C. A preferred temperature range is from about 10 to about 40C.The hydrolysis reaction is suitably performed in an aqueous medium. Anorganic co-solvent such as a lower alkanol or an organic ether may beemployed if desired. At the completion of the reaction, the reactionmixture is acidified to free the carboxylic acid reaction product forthe purposes of isolation.

Compounds of formula III are depicted as existing in both the diketo andthe enolized form. It should be understood that the ratio of tautomersof the compound can vary depending upon solvent, temperature, pH, and soforth. In the experimental section and the claims, the compounds offormula III are named as the enol form for convenience only.

CO I'I wherein R and R are as above; R, and R taken independently areeach lower alkyl; and R, and R taken together with the nitrogen atomform a 5- or 6-membered saturated heterocyclic ring having at the mostone additional heteroatom selected from the group consisting of nitrogenand oxygen.

The kcto carboxylic acid of formula III may be converted to thefluorogen of formula I by a two step procedure. In the first step theketo acid of formula III is reacted with an amino-methenylating agent toafford an anamine of formula IV. Suitable aminomethenylating agentsinclude acetals of an N,N-disubstituted formamide, e.g.dimethylformamide dimethylacetal; tris(secondary amino)methanes, e.g.tris(dimethylamino)methane and tris(piperidino) methane; andbis(secondary amino)lower alkoxy methanes, e.g. bis(dimethylamino)-t-butoxy methane.

The amino moiety tris(secondary amino)methanes have the general formulaR -c N\R2 and bis(secondary amino)lower alkoxymethanes have the generalformula Examples of amino moieties inc ude those where R, and R eachtaken independently are lower alkyl, e.g. dimethylamino anddiethylamino; and those where R and R taken together with the nitrogenform a 5-6-membered heterocyclic ring, e.g. piperidino, morpholino,pyrrolidino, and so forth. This reaction may be carried out in any inertorganic solvent. Particularly preferred solvents include formamides,especially dimethylformamide. An excess of aminomethenylating agent maybe also utilized as solvent. The preparation of the enamine may beeffectuated over a temperature range of from about 0 to about although atemperature range of from about 10 to about 40 is preferred. Atemperature of about room temperature is especially preferred.

The enamine offormula IV may be then directly converted to the fluorogenof formula I by aqueous hydrolysis at a neutral acidic or basic pH.Thus, an acid such as mineral acid, e.g. HCl; or a weak base as analkali metal bicarbonate, e.g. sodium bicarbonate, may be present in thehydrolysis medium. In general, it is preferred to effectuate theaforementioned hydrolysis in the presence of a weak base such as analkali metal bi-' carbonate. Upon completion of the hydrolysis andacidification of the reaction medium (in cases where the hydrolysis iscarried out on the neutral or basic side) the desired cyclized fluorogenof formula I is obtained. The temperature of the hydrolysis reaction issuitably in the range of from about 0 to about 100, although atemperature of about 10 to 40 is preferred. A temperature of abou'f roomtemperature is especially preferred. The conversions II III, III IV andIV I may be carried out with isolation and purification of the productat each stage or, preferably, can be carried out with isolation andpurification only of the final fluorogen of formula I.

The fluorogens of formula I react readily with primary aminecontainingcompounds, particularly with peptides, amino acids and biogenic amines,e.g. catecholamines, to form highly fluorescent materials. Thus, thecompounds of formula I serve as highly sensitive reagents for thedetection of primary amine-containing compounds. Since the compounds offormula I are stable to aqueous media, the detection of primaryaminecontaining compounds present in said media is rendered highlypractical. This is especially important for the detection of amino acidsand peptides which are generally found and analyzed in aqueous media.

The reaction between fluorogens of formula I and primaryamine-containing compounds is a rapid and quantitative one. The reactionmay be carried out over a wide pH range of from about pH 4 to about pH ll.

However, the rate and completeness of the reaction of the fluorogen withthe primary amine-containing compounds is optimal within a pH rangebetween about 8 and 9. Thus, reaction of a compound of formula I with asimple primary amine, such as ethylamine, is instantaneously complete atroom temperature within this pH range.

It is immediately apparent that the use of a fluorogenic reagent offormula I for the detection of primary amine-containing compounds issuperior to the prior art technique hereinabove described involving aternary reaction between a primary aminecontaining compound, ninhydrinand phenylacetaldehyde. A further superiority of the reagents andmethods of the present invention is seen from the fact that, in theprior art procedure, it was necessary to heat the mixture of reagents toa temperature of about 60C. for to 30 minutes to develop maximumfluorescence, whereas, in contradistinction, the reagent of the presentinvention develops maximum fluorescence instantaneously at roomtemperature. The fluorescence obtained using equal concentrations ofprimary amine-containing compounds utilizing the reagents of the presentinvention is generally in the range of 10 to 50 times greater than thatobtained by using the prior art technique. Thus, the reagents ofthepresent invention may be utilized to detect exceedingly minutequantities ofprimary amineeontaining compounds which was heretoforeimpossible. Due to the sensitivity ofthe reagents of the presentinvention, small amounts of peptides and amino acids can be detected.For example, one can detect and examine quantitites of biologicallyactive peptides isolated from a given organ or a single laboratoryanimal.

The use of the reagents of the present invention for the detection ofprimary amine-containing compounds may be illustrated in a number ofways. For example, the presence of primary aminecontaining compounds insolution may be determined by mixing the sample,

with an excess of fluorogen of formula 1 at a desired pH, and measuringthe fluorescence. It is preferred to have the primary amine-containingcompound in solution in an aqueous medium and to treat it with asolution of the fluorogen in a non-hydroxylic, water miscible solventsuch as acetonitrile, dimethylformamide, dimethylsulfoxide,tetrahydrofuran, dioxane; and the like. A l0-fold to 5,000-fold molarexcess of fluorogen is preferred. A 50-fold to 1,000-fo1d molar excessis especially preferred.

Alternatively, the fluorogens of the present invention may be utilizedto detect the presence of primary amine-containing compounds, especiallyamino acids, on paper or thin-layer chromatographic systems. In such atechnique, the reagent in a solvent, preferably a volatile organicsolvent, optionally containing a pH buffer, is applied to thechromatographic system, usually as a spray, and the paper or thin-layerchromato: gram is examined under a fluorescent light source. Thedevelopment of fluorescence is instantaneous at room temperature and,thus, there is no need to heat thechromatogram to develop fluorescence.This offers a decided advantage over prior art techniques.

Another application of the fluorogens of the present invention is theuse in automated analysis of amino acids. In such a procedure, a samplestream which is a portion of the effluent of a chromatographic column ismixed with the fluorogenic reagent of formula 1 at the desired pH andthe mixture immediately analyzed by a spectrofluorometer. in thismanner, a monitoring of the fluorescence exhibited by a continuoussample stream is indicative of the presence and concentration of variousamino acids.

A further appreciation of the preparation of the novel fluorogens of thepresent invention and their use in producing highly fluorescentsubstances upon reaction with primary aminecontaining compounds may beobtained from the following examples.

EXAMPLE 1 A solution of 8.1 g., 1,4-isochromandione (0.5 mole), 6.0 g.benzaldehyde (0.055 mole) and 0.1 ml. piperidine in ml. benzene washeated at reflux temperature under nitrogen until the theoretical amountof water (0.05 mole) had collected in a Dean-Stark trap (ca. 2 hrs.).The reaction mixture was then cooled to 10 and the product was filteredoff. The filter cake was recrystallized from benzene to afford 11.4 g.,3- benzylidene-4-keto-3,4-dihydroisocoumarin, m.p.

Anal. Calcd. for C H O C, 76.79; H, 4.03

Found: C, 76.49; H, 4.05.

Analogously were prepared from 1,4-chromandione and the correspondingaldehydes the compounds listed i Tab e. I;

li il R'= Empirical Analysis mp I Formula Calcd. Found 2-methoxyphenyl200 C H O, C,72.85 72.96 H, 4.32 4.19 3-methoxyphcnyl 153 C H O, C,72.8572.52 H, 4.32 4.21 4-mcthoxyphenyl 200 C,,H,,O C,72.85 72.56 H,'4.3Z4.29 2,4-dimethoxyphenyl 201 C H O C,69.67 69.53 H, 4.55 4.57

Table I Continued R'= Empirical Analysis 4 mp Formula Cale Found2,5-dimethoxyphenyl 160 C H O C,69.67 69.65 H. 4.55 4.543,5-dimethoxyphenyl 178 C H O C,69.67 69.62 H, 4.55 4.633,4,5-trimethoxyphcnyl 144 C,,,H ,O,, C,67.05 66.71 H, 4.75 4.562,4,5-trimethoxyphenyl 210 C H O C,67.05 67.35 H, 4.75 4.773,4-methylenedioxyphenyl 226 C,,H,,.O,-, C,69.39 69.16 H, 3.43 3.333-chloropheny1 224 C H CIO C,67.50 67.41 H, 3.19 3.2i 4-chlorophenyl 203C,,,H,,C10 C,67.50 67.26 H, 3.19 3.04 4-bromopheny1 204 C,,.H,Br0,.C,58.38 58.46 H, 2.76 2.66 3-indolyl 308 C H NO C,74.73 74.69 H, 3.833.94 N, 4.84 4.92 Z-naphthyl 251 C H O C,79.99 80.28 H, 4.03 4.01lnaphthyl 184 C H O C,79.99 80.27

H, 4.03 3.95 n-propyl 58 c n o, C,72.21 72.01 H, 5.59 5.44

EXAMPLE 2 Table II- Contrnued To a solution of g.3-benzylidene-4-keto-3,4-

dihydroisocoumarin in 400 ml. methanol were added 100 m1. 1N aqueoussodium hydroxide. The mixture was stirred at room temperature forminutes. It was then diluted with water and acidified with 10%hydrochloric acid. The precipitating o-(ahydroxycinnamoyl) benzoic acidwas extracted twice with chloroform. The organic extracts were washedwith water, dried over sodium sulfate and evaporated under reducedpressure to dryness. The residue was dissolved in 80 ml.dimethylformamide. The solution was cooled to 0, and, while stirring, 24ml. tris(dimethylamino)methane were added. Stirring was con'tinued atroom temperature for 60 minutes. Theldimethylamino-2-phenyl-4(o-carboxyphenyU-l-buten- 3,4-dione which hadformed was not isolated, but rather, the DMF-solution was poured intoice-water. The resultant aqueous alkaline solution was extracted withether. The ether extract was discarded and the aqueous layer wasacidified with dilute hydrochloric acid. The desired4-phenylspiro[furan;2(3H)-1- -phthalan]3,3-dione was extracted 3 threetimes with ether/benzene 1:1 The combined extracts were dried oversodium sulfate and evaporated under reduced pressure. The crystallineresidue was resuspended in ether and filtered off to give 12.0 G. purematerial, mp 153.

, Anal. Calcd. for C, H,.,O C l;

73.3 3. Found: C, 73.4

The compounds listed in Table 11 were prepared by analogous procedures,starting with the appropriate 3-' arylideneor 3-alkylidene-4-keto-3,4-

dihydroisocoumarins, prepared as in Example 1 Table II EmpiricalAnalysis R'= mp Formula Calcd. Found 2-methuxyphenyl 153 C H Q, C,70.1370.24 H, 3.92 3.86 3-methoxyphenyl 1 18 C H O, C,70.13 70.13 H, 3.923.71 4-methoxyphenyl 158 C H O, C70. 13 70.40

H. 3.92 4.01 2,4-dimethoxyphenyl 133 C H O C,67.45 67.21 H, 4.17 4,062,5-dimethoxyphenyl 148 C H O C,67.45 67.54 H, 4.17 4.093,5-dimethoxyphenyl 138 C H O 6;.72 3,4,5-trimethox hen 1 205 C H.,,(),C, 65.21 65.33 y 11.4.38 4.26 2,4,5-trimethox henl 179 C H O C,65.2165.34 YP 10 m 1 H'4-38 4'2; 3,4-meth lenediox hen 1 195 C,,H ,O,,C,67.08 67.0

y W y H, 3.13 3.43 3-chlorophenyl 144 C,,H,,C1O, C,65.3() 65.32 H. 2.902.61 4-chlorophenyl 167 C,,H,,C1O C.65.30 65.10 H. 2.90. 2.924-bromopheny1 181 C,,H,,Br0 Q57. 17 57.25

H, 2.54 2.63 3-indolyl 186 C H NO C,71.92 72.16 H, 3.49 3.43 N, 4.414.16 Z-naphthyl 170 C. H,,o, C.76.82 76.62 H. 3.68 3.58 l-naphthyl 191 ct-1, 0, c.7682 76.85 H, 3.68 3.53

n-propyl oil C ll O M LE 1 To a suspension of 2.5 g.3-benzylidene-4-keto-3,4-

dihydroisocoumarin in 50 ml. methanol were added 10 ml. of 1N aqueoussodium hydroxide. The mixture was stirred at room temperature for 1hour. A dark red solution resulted. The solution was diluted with water,

acidified with 10 m1. of 1N hydrochloric acid and ex- 'room temperature.Petroleum ether was added to the solution to the point of incipientturbidity. The resulting mixture was kept at room temperature for 16hours, after which time the desired acid had crystallized and wasfiltered off. The crystals were washed on the filter ,'with methylenechloride/petroleum ether (1:9) and dried in vacuo to afford 2.0 g. ofo-(a-hydroxycin namoyl)benzoic acid, mp 99105.

The compounds listed in Table 111 were prepared by analogous procedures,starting with the appropriate 3-arylidene-4-keto-3,4-dihydroisocoumarins, prepared asphenylspiro[furan-2( 3H l '-phthalan 13,3 '-dione.

Table III Empirical Analysis R'= mp Formula Calcd. Found 2-methoxyphenyll3l-l 36 C l-l 0 0,68. 68. 1 4 C, 4.73 4.73

3-methoxyphenyl l33-l35 C H O 0,68. 45 68. 75 7 5 n, 4.73 5.00

l-methoxyphenyl ll7-l23 C H O C 68.45 68.52 17 5 H: 0.73 4.78

2,5-dimethoxyphenyl l31-l32 C H O 0,65 .85 65. 82 H, 3.91 5.20

3,5-dimethoxyphenyl 131-13 C H O C,65 85 66.08 H, 4.91 5 .03

2, i,5-trimethoxyphenyl 152-165 0 a o C,63.68 63.33 9 7 a, 5.00 5.11

3-chlorophenyl ll7-l18 C ll ClO C,63. 48 63.59

t-bromophenyl' l30-l32 C H BrO C,55.35 55.37 16 11 l H 3.19 3.2 1

B-indolyl l20-l25 C H NO 2 C 63. 52 63. 63 18 13 H: 0.03 3.78 N 4 .01 3.9n

l-napiithyl l5 l-l60 C H O C 75 U6 T5 .5 H: 0. 4 4.61

EXAMPLE 4 55 EXAMPLE 5 To a solution of 268 mg. o-(a-hydroxycinnamoyl)-benzoic acid in 5 ml. of dimethylformamide were added 2 ml.dimethylformamide dimethyl acetal. The reaction mixture was kept at roomtemperature for 17 hours. The solution containing l-dimethylamino-2-phenyl-4(o-carboxyphenyl)-l-buten-3,4-dione was then poured into water,acidified with dilute hydrochloric acid and extracted with methylenechloride. The organic extract was dried over sodium sulfate andconcentrated in vacuo. The concentrate was applied to a short silica gelcolumn (4.5 g.) and eluted with methylene chloride. The eluate wasevaporated in vacuo and the residue was was recrystallized frommethylene chloride/ether. There were obtained llO mg. 4-

and evaporated in vacuo. the residue containingo-(a-hydroxycinnamoyl)benzoic acid was dissolved in -l5O ml.dimethylformamide. To this solution were added 30 g.tris-(piperidinolmethane. The mixture was stirred at room temperaturefor 5 hours and the solution containing l-( l-piperidino)-2-phenyl-4-(ocarboxyphenyl)-l-buten-3,4-dione then poured into ice-water. Thealkaline aqueous mixture was extracted with ether. The ether extract wasdiscarded. The aqueous layer was acidified with 1N hydrochloric acid(300 ml.) and extracted 4 times with methylene chloride. The organicextracts were combined, washed with water, dried over sodium sulfate andevaporated to dryness under reduced pressure. The residue was dissolvedin 75 ml. methylene chloride and treated with l g. of Norite at refluxtemperature. The hot solution was tiltered. The filtrate wasconcentrated to 50 ml. and then diluted with 100 ml. ether. From themixture there crystallized 8.4 g. of 4-phenylspiro[furan-2(3H),lphthalan]-3,3'-dione; mp l5ll53.

The compounds listed in Table II were prepared by analogous procedures,starting with the appropriate 3- arylideneor 3-alkylidene-4-keto-3,4-dihydroisocumarins prepared as in Example 1.

EXAMPLE 6 To a solution of 4.47 g. o-(a-hydroxy-Z- methoxycinnamoyl)benzoic acid in 30 ml. dimethylformamide were added 5 g.tris(dimethylamino)- methane. The mixture was stirred at roomtemperature for 3% hours. Most of the excess formulating agent and thesolvent were then removed under reduced pressure at 5560. The residuecontaining l-dimethylamino-2-(o-methoxyphenyl)-4-(o-carboxylphenyl)-l-buten-3,4- dione wasredissolved in 10 ml. of dimethylformamide and this solution added to100 ml. water. The resulting alkaline mixture was extracted withmethylene chloride. The organic extract was discarded. The aqueous layerwas acidified with dilute hydrochloric acid and extracted twice withmethylene chloride. The organic phases were combined, washed with water,dried over sodium sulfate and evaporated to dryness under reducedpressure. The remaining oil wasdissolved in ether. Crystals precipitatedfrom the ether solution' upon refrigeration. They were collected byfiltration to afford 2.4 g. 4-(2-methoxyphenyl) spirolfuran-2(3H),l-phthalan]-3,3'-dione, mp l52-l54.

The compounds listed in Table III were converted to the correspondingspirolactones listed in Table I] by analogous procedures.

EXAMPLE 7 Fluorometric Assay Procedure:

Place in a test tube:

a. l ml. of peptide solution nanomole leucylalanine in 1 ml, pH8-phosphate buffer) and b. 2 ml. pH 8-buffer solution (Fisher). Whileagitating with a vibro-mixer add rapidly:

c. 1 ml. of reagent solution (containing 0.2 millimole of spirolactoneof Table ll in 100 ml. acetonitrile).

Final concentrations:

Leu-ala: 5 nanomole/ml reagent: ().5 umole/ml 100 fold excess)acetonitrile:

aqueous buffer: 75% Measure fluorescence immediately. lnstrumcntion:

Farrand Spectrol'luorometer Excitation: 390 nm; slits l0,l0

Emission: 385 nm; slits 20,20

Sensitivity l g I Results:

With 4-phenylspiro[furan-2(3H),l'-phthalan]3.3- dione as the reagent afluorescent intensity of 80. relative to a fluorescence intensity of (atthe same excitation and emission settings) for a quinine standard, 10mg/ml 0.01N H 50 was observed.

The above experiment was repeated using ethylamine in place ofleucylalanine, and ethanol in place of acetonitrile. The fluorescenceintensity obtained by utilizing various spirolactone reagents of Tablell is shown in Table IV. The fluorescence intensity obtained using4-pheny|spiro[furan-2( 3H l -phthalan 13,3 -dione has been arbitrarilyassigned the value I00, and theother intensities are given relative toit.

Starting with the appropriate 5-substituted-2-acetylbenzoic acids, andproceeding, first according to known procedures, to the corresponding7-substituted- 1,4-isochromandione, and then, according to the procedureof Example 1, to the 7-substituted-3-benzylidene-4-keto-3,4-dihydroisocoumarin, the fol lowing series ofintermediates and fluorogens were prepared, following the procedures ofExample 2:

3-benzylidene-4-keto-7-methyl-3,4- dihydroisocoumarin,

2(a-hydroxycinnamoyo)-5-methyl-benzoic acid,l-dimethylamino-2-phenyl-4(2-carboxy-4-methylphenyl)-l-buten-3,4-dione,,

5'-methyl-4-phenylspiro[furan-2(3H ),l -phthalan 3,3-dione,

3-benzylidene-4-keto-7-butyl-3,4- dihydroisocoumarin,

2-(a-hydroxycinnamoyl)-5-butyl-benzoic acid,l-dimethylamino-2-phenyl-4(2-carboxy-4-butylphenyl)-l-buten-3,4-dione,

5-butyl-4-phenylspiro[furan-2( 3H),] '-phthalan]-' 3,3-dione,

3-benzylidene-4-keto-7-methoxy-3,4- dihydroisocoumarin,

Z-(a-hydroxycinnamoyl)-5-methoxy-benzoic acid,

1-dimethylamino-2-phenyl-4(2-carboxy-4-methoxyphenyl )-l-buten-3,4-dione,

5'-methoxy-4-phenylspiro[furan-2( 3H),] phthalan]-3,3-dione,

3-benzylidene-4-keto-7-chloro-3,4- dihydroisocoumarin,

2-(a-hydroxycinnamoyl)-5-chloro-benzoic acid,

l-tlimcthylamino-2-phenyl-4(2-carboxy-4-chlorophenyl )-l-buten-3,4-dione,

5-chloro-4-phenylspirolfuran-2(3H),l 3,3'-dione.

What is claimed is:

l. A method for determining a primary amine- '-phthalan containingcompound in a mixture containing same which method comprises:

treating said mixture with a compound offormula wherein R is hydrogen,halogen, lower alkyl or lower alkoxy and R is lower alkyl or aryl toproduce a fluorescent substance therein and fluo rescing said substance.

2. The method of claim 1 wherein the primary aminecontaining compound ispresent on at least a portion of a surface of a paper or thin-layerchromatographic medium and the compound of formula I is applied to saidsurface of said chromatographic medium.

3. The method of claim 2 wherein R is hydrogen and R is phenyl.

4. The method of claim 3 wherein the pH of the chromatographic medium iskept between 8 and 9.

5. The method of claim 3 wherein the compound of formula I is present ina molar excess of between 10 and 5000 as compared with the amount ofprimary amine-containing compound.

6. The method ofclaim 3 wherein the primary aminecontaining compound isan a-amino acid.

7. The method ofclaim 3 wherein the primary aminecontaining compound isa peptide.

8. The method of claim 1 wherein the primary amine containing compoundis determined by an automated procedure which comprises:

a. continuously treating a sample stream containing said primaryamine-containing compound in solution with an excess of a compound offormula I, and

b. passing the mixture obtained in step (a) at a constant rate through aflow cell ofa fluorophotometer whereby any fluorescent material presentis fluoresced and detected.

9. The method of claim 8 wherein the sample stream is at least a portionofthe effluent ofa chromatographic column.

10. The method of claim 8 wherein R is hydrogen and R is phenyl.

11. The method of claim 8 wherein the compound of formula I is presentin a molar excess of between l0 and 5000 as compared with the amount ofprimary amine-containing compound.

12. The method of claim 8 wherein the pH of the solution beingfluoresced is maintained between 8 and 9.

13. The method of claim 8 wherein the primary amine-containing compoundis an a-amino acid.

14. The method of claim 8 wherein the primary amine-containing compoundis a peptide.

15. The method of claim 8 wherein the total amount of a specific knownprimary amine-containing compound present in the sample stream isquantitatively determined by a. determining the total fluorescenceproduced by a known quantity of said primary amine-containing compound,

b. determining the total fluorescence produced by the unknown quantityof said primary aminecontaining compound under the same conditions asstep (a), and

c. obtaining the unknown amount by multiplying the known quantity by theratio of the unknown to the known total fluorescence. v

16. The method of claim 1 wherein said treatment is done in solution.

17. The method of claim 16 wherein R is hydrogen and R is phenyl.

18. The method ofclaim 16 wherein the pH of the solution is between 8and 9.

19. The method of claim 16 wherein the compound of formula I is presentin a molar excess of between 10 and 5000 as compared with the amount ofprimary amine-containing compound.

20. The method of claim 16 wherein the primary amine-containing compoundis an a-amino acid.

21. The method of claim 16 wherein the primary amine-containing compoundis a peptide.

22. The method ofclaim 16 wherein a fluorophotometer is utilized forfluorescing said substance.

23. The method of claim 16 wherein the concentration in solution of aknown primary amine-containing compound is quantitatively determined bya. determining the fluorescence produced by a measured concentration ofsaid amine,

b. determining the fluorescence produced by the unknown concentration ofsaid amine, and

c. obtaining the unknown concentration by multiplying the knownconcentration by the ratio of the unknown to the known fluorescenceyalues

1. A METHOD FOR DETERMINING A PRIMARY AMINE-CONTAINING COMPOUND IN AMIXTURE CONTAINING SAME WHICH METHOD COMPRISES: TREATING SAID MIXTUREWITH A COMPOUND OF FORMULA I
 2. The method of claim 1 wherein theprimary amine-containing compound is present on at least a portion of asurface of a paper or thin-layer chromatographic medium and the compoundof formula I is applied to said surface of said chromatographic medium.3. The method of claim 2 wherein R is hydrogen and R'' is phenyl.
 4. Themethod of claim 3 wherein the pH of the chromatographic medium is keptbetween 8 and
 9. 5. The method of claim 3 wherein the compound offormula I is present in a molar excess of between 10 and 5000 ascompared with the amount of primary amine-containing compound.
 6. Themethod of claim 3 wherein the primary amine-containing compound is anAlpha -amino acid.
 7. The method of claim 3 wherein the primaryamine-containing compound is a peptide.
 8. The method of claim 1 whereinthe primary amine containing compound is determined by an automatedprocedure which comprises: a. continuously treating a sample streamcontaining said primary amine-containing compound in solution with anexcess of a compound of formula I, and b. passing the mixture obtainedin step (a) at a constant rate through a flow cell of a fluorophotometerwhereby any fluorescent material present is fluoresced and detected. 9.The method of claim 8 wherein the sample stream is at least a portion ofthe effluent of a chromatographic column.
 10. The method of claim 8wherein R is hydrogen and R'' is phenyl.
 11. The method of claim 8wherein the compound of formula I is present in a molar excess ofbetween 10 and 5000 as compared with the amount of primaryamine-containing compound.
 12. The method of claim 8 wherein the pH ofthe solution being fluoresced is maintained between 8 and
 9. 13. Themethod of claim 8 wherein the primary amine-containing compound is anAlpha -amino acid.
 14. The method of claim 8 wherein the primaryamine-containing compound is a peptide.
 15. The method of claim 8wherein the total amount of a specific known primary amine-containingcompound present in the sample stream is quantitatively determined by a.determining the total fluorescence produced by a known quantity of saidprimary amine-containing compound, b. determining the total fluorescenceproduced by the unknown quantity of said primary amine-containingcompound under the same conditions as step (a), and c. obtaining theunknown amount by multiplying the known quantity by the ratio of theunknown to the known total fluorescence.
 16. The method of claim 1wherein said treatment is done in solution.
 17. The method of claim 16wherein R is hydrogen and R'' is phenyl.
 18. The method of claim 16wherein the pH of the solution is between 8 and
 9. 19. The method ofclaim 16 wherein the compound of formula I is present in a molar excessof between 10 and 5000 as compared with the amount of primaryamine-containing compound.
 20. The method of claim 16 wherein theprimary amine-contAining compound is an Alpha -amino acid.
 21. Themethod of claim 16 wherein the primary amine-containing compound is apeptide.
 22. The method of claim 16 wherein a fluorophotometer isutilized for fluorescing said substance.
 23. The method of claim 16wherein the concentration in solution of a known primaryamine-containing compound is quantitatively determined by a. determiningthe fluorescence produced by a measured concentration of said amine, b.determining the fluorescence produced by the unknown concentration ofsaid amine, and c. obtaining the unknown concentration by multiplyingthe known concentration by the ratio of the unknown to the knownfluorescence values.